Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Gascoigne, Karen E., and Iain M. Cheeseman. “Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.” Chromosome Research 21, no. 4 (July 22, 2013): 407-418. As Published http://dx.doi.org/10.1007/s10577-013-9368-6 Publisher Springer-Verlag Version Final published version Citable link http://hdl.handle.net/1721.1/82625 Detailed Terms http://creativecommons.org/licenses/by/3.0/ Chromosome Res (2013) 21:407–418 DOI 10.1007/s10577-013-9368-6 Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis Karen E. Gascoigne & Iain M. Cheeseman Received: 26 April 2013 /Revised: 30 May 2013 /Accepted: 9 June 2013 /Published online: 22 June 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Chromosomal rearrangements can radically Our results suggest that a single dicentric chromosome alter gene products and their function, driving tumor could contribute to tumor initiation. formation or progression. However, the molecular origins and evolution of such rearrangements are varied Keywords Chromosome . Dicentric . Centromere . and poorly understood, with cancer cells often con- Kinetochore . Mitosis . Translocation . Genomic taining multiple, complex rearrangements. One mech- rearrangement . Transformation . Cancer anism that can lead to genomic rearrangements is the formation of a “dicentric” chromosome containing Abbreviations two functional centromeres. Indeed, such dicentric CML Chronic myeloid leukemia chromosomes have been observed in cancer cells. MLL Mixed lineage leukemia Here, we tested the ability of a single dicentric chro- AML Acute myeloid leukemia mosome to contribute to genomic instability and neo- DNA Deoxyribonucleic acid plastic conversion in vertebrate cells. We developed a GFP Green fluorescence protein system to transiently and reversibly induce dicentric LacI Lac repressor chromosome formation on a single chromosome with lacO Lac operator high temporal control. We find that induced dicentric IPTG Isopropyl β-D-1-thiogalactopyranoside chromosomes are frequently damaged and mis- p-H2AX Phospho-histone-H2AX segregated during mitosis, and that this leads to exten- 53BP1 53 binding protein 1 sive chromosomal rearrangements including translo- FISH Fluorescence in situ hybridization cations with other chromosomes. Populations of pre- SKY Spectral karyotype neoplastic cells in which a single dicentric chromo- some is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Introduction Abnormal chromosome and nuclear morphology are Responsible Editor: Beth A. Sullivan key diagnostic characteristics of tumor cells (Hanahan and Weinberg 2000). Almost all cancers show an abnor- K. E. Gascoigne : I. M. Cheeseman Whitehead Institute for Biomedical Research, mal and often unstable karyotype, with aneuploid chro- Nine Cambridge Center, Cambridge, MA 02142, USA mosome numbers and the presence of genomic rearrangements including translocations, deletions, * I. M. Cheeseman ( ) fusions, duplications, and insertions. Such rearrangements Department of Biology, Massachusetts Institute of Technology, Cambridge, USA can radically alter gene products and function, driving e-mail: [email protected] tumor formation or progression. For example, the 408 K.E. Gascoigne, I.M. Cheeseman Philadelphia chromosome translocation between chromo- could also result in the formation of new dicentric somes 9 and 22 produces the Bcr-Abl fusion protein found fusion chromosomes. This process of cascading chro- in most CMLs and is sufficient to drive the disease mosomal damage has been termed the “breakage– (Ben-Neriah et al. 1986). Similarly, the MLL transcrip- fusion–bridge cycle” and is strongly associated with tion factor is commonly found as a fusion protein with cancer initiation (Cosme-Blanco and Chang 2008; multiple partners in AML and is a strong predictor of McClintock 1939). Although it has been hypothesized poor prognosis (Ayton and Cleary 2001). The origins that rearrangements stemming from such cycles are and evolution of such chromosome rearrangements are induced by errors in mitosis, this mechanism has not varied and poorly understood, and cancer cells often been directly tested at the molecular level in verte- contain multiple, complex rearrangements. Previous brate cells. In addition, the extent to which a single work has proposed several potential mechanisms for dicentric chromosome could lead to complex genomic such rearrangements including errors in replication and rearrangements and neoplastic transformation has not recombination (Colnaghi et al. 2011), errors in cytoki- been explored. nesis that result in tetraploidy (Fujiwara et al. 2005), Here, we generated an experimental system to test the errors in centrosome number and function that cause effects of assembling two kinetochores on a single chro- spindle defects (Ganem et al. 2009), telomere erosion mosome on genome stability and cellular transformation. (Hemann et al. 2001), and chromosome “shattering” via Our system makes it possible to transiently induce an chromothripsis (Stephens et al. 2011). ectopic kinetochore-like structure at a non-centromeric An additional mechanism that can lead to genomic site on a single chromosome that also contains an rearrangements is the formation of a dicentric endogenous centromere, thereby generating a function- chromosome—a chromosome containing two func- ally dicentric chromosome. We demonstrate that induc- tional centromeres. Indeed, such dicentric chromo- tion of a single dicentric chromosome results in chro- somes have been observed in cancer cells, particularly mosome mis-segregation and DNA damage. In subse- hematological malignancies (Gisselsson et al. 2000; quent cell divisions, we find that induction of the dicen- Mackinnon and Campbell 2011; Yamamoto et al. tric chromosome and the corresponding DNA damage 2011). Such cells often display extensive and complex results in genomic rearrangements similar to those genome rearrangements. However, the causal link observed following telomere erosion-induced break– between the presence of a dicentric chromosome and bridge–fusion cycles. The presence of the dicentric other genome alterations remains unclear. As the cen- chromosome also leads to global genome instability tromere provides the site of kinetochore formation and and karyotype abnormalities similar to those found in attachment to the microtubule-based mitotic spindle, it cancer cells. Finally, induction of the dicentric chromo- is essential that only one functional centromere exist some in a population of cells leads to the acquisition of per chromosome. In rare cases, dicentric chromo- characteristics of cellular transformation, suggesting somes can be stably maintained by inactivating one that the presence of a single dicentric chromosome of the centromeres, but the mechanisms of this inacti- could contribute to tumor initiation. vation remain poorly understood, occur at low fre- quency, and are likely to be stochastic (reviewed in Stimpson and Sullivan 2010). However, in many cases, both centromeres remain active and act as building sites Materials and methods for kinetochores during mitosis. The presence of multi- ple kinetochores on a single chromosome could result in Cell culture and transfection a single sister chromatid simultaneously attaching to both spindle poles, which would cause chromosome Cell lines were maintained as described previously mis-segregation or deoxyribonucleic acid (DNA) dam- (Kline et al. 2006). NIH3T3 lac operator (lacO) cells age. Spindle-based forces could also generate chro- (a generous gift from T. Mistelli; Soutoglou et al. mosome breaks such that a dicentric chromosome 2007) were maintained in 400 μg/ml Hygromycin B. would be fragmented during mitosis. Chromosome Stable clonal cell lines expressing green fluorescence breaks, in turn, often lead to chromosome fusions that protein (GFP)-Lac repressor (LacI) fusions were generated alter or dysregulate genes around the break site and as described previously and maintained in 10 mM Induced dicentric chromosome formation 409 isopropyl β-D-1-thiogalactopyranoside (IPTG) (Gascoigne indicated. Cells were incubated at 37 °C and colonies et al. 2011). formed after 4 weeks were scored. Invasion assays were carried out in 24-well Transwell® chamber plates con- Immunofluorescence and microscopy taining a Cultrex® basement membrane-coated polycar- bonate support with 8 μm pores (Corning). The cells Immunofluorescence was conducted as described previ- were cultured for 14 days in the presence or absence of ously (Kline et al. 2006) using anti-phospho-histone 10 mM IPTG as indicated, then seeded into the top H2AX (Millipore) and 53 binding protein 1 (Novus) chamber at a density of 100,000 cells per chamber. antibodies. Cy2, Cy3, and Cy5-conjugated secondary After incubation at 37 °C for the indicated time periods, antibodies were obtained from Jackson Laboratories. cells were manually removed from the top chamber and DNA was visualized using 10 μg/ml Hoechst. Images wells were fixed for 10 min in 4 % formaldehyde before were acquired on a DeltaVision Core deconvolution staining with 0.1 % crystal violet solution. Cells retained microscope (Applied